Studies were continued on the separation of glutamine synthetase (GS) species that differ in the number (from 0 to 12) and distribution of adenylylated subunits. Some of the difficulties encountered previously in the use of affinity chromatography on Affi-blue sepharose are likely due to (1) variations in the ambient temperature at which the chromatography was performed; (2) heterogeniety in the affinity of Cibarcron dye-sepharose complexes that interact with GS; (3) variations in the ratio of ADP to Mn++ that occur during the gradient elutions; and (4) the presence of isomeric forms of GS containing identical numbers of adenylylated subunits. The differential precipitability of GS species containing on the average nearly the same number of adenylylated subunits by means of anti-AMP antibodies is also likely due to the presence of isomeric enzyme forms containing the same number of adenylylated subunits.